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The therapeutic approach to thymic cysts remains a subject of controversy. Predicted biomarkers for identifying thymic cysts and thymoma (THYM) are crucial. In this research, patients diagnosed with thymic cysts (MTC, n = 6) and thymoma (B1, n = 6; B3, n = 6) were enrolled. Proteins of superior quality were subjected to TMT labeling and UPLC-MS, and differentially expressed proteins (DEPs) were identified. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interactive network analyses were applied to the DEPs. Some key differentially expressed genes(DEGs) were corroborated through GEPIA 32. The pan-cancer expression levels of key DEGs remarkably linked with prognosis were determined utilizing The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN). Eventually, 49 DEPs were identified in the B1 vs. MTC comparison (17 upregulated and 32 downregulated), 27 in the B3 vs. MTC comparison (8 upregulated and 19 downregulated), and 38 in the B3 vs. B1 comparison (9 upregulated and 29 downregulated). IL13RA1 (down), galectin-3 binding protein (LGALS3BP)(up), PRCSH (down), C3 (down), MXRA5 (down), TNN (down), CFHR1 (down), SUN3 (down) were jointly altered in both B1 vs. NZ and B3 vs. NZ. GEPIA validated that LGALS3BP was significantly upregulated in thymoma patients. In conclusion, LGALS3BP might be an essential biomarker to identify thymoma from the thymic cyst., (© 2024. The Author(s).)

Aims: Thymic carcinoma and atypical thymoma (WHO type B3 thymoma) are unusual tumours the separation of which may be challenging in small biopsies. Both tumours consist of epithelioid tumour cells that share similar morphology and immunophenotype with conventional markers. Therefore, additional antibodies are needed to differentiate between these tumours., Methods: For this purpose, a panel of immunohistochemical stains including PAX2, PAX5, PAX8 (all monoclonal) and CD70 was used on whole tumour sections of 30 thymic carcinomas and 30 atypical thymomas to determine the expression pattern of these antibodies. In addition, all tumours were stained with markers that are well known to be expressed in both tumours, including pancytokeratin and cytokeratin 5/6. The percentage of positive tumour cells as well as the intensity of staining were evaluated and scored., Results: PAX5 stained close to 70% of thymic carcinomas while all atypical thymomas were negative for this marker. CD70 was expressed in 18 thymic carcinomas (60%) and in 1 case of atypical thymoma (3%). On the other hand, monoclonal PAX8 was negative in all cases while PAX2 was positive in a single thymic carcinoma. Of the established stains, pancytokeratin and cytokeratin 5/6 were equally positive in both tumours., Conclusions: Among the markers explored, only PAX5 and CD70 appear to be differentially expressed and are predominantly restricted to thymic carcinomas. Therefore, in small biopsy specimens and in resections in which the morphological features remain equivocal, application of these particular stains may facilitate separation of thymic carcinoma and atypical thymoma., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Metaplastic thymoma (MT), a rare subtype of thymic epithelial tumors (TETs), harbors YAP1::MAML2 fusions. Poroma, a skin tumor, also carries these fusions and exhibits a unique staining pattern for YAP1 immunohistochemistry (IHC), namely, a YAP1 N-terminus (YAP1[N])-positive but YAP1 C-terminus (YAP1[C])-negative pattern. In this context, MT was recently reported to lack YAP1(C) expression exclusively among TET subtypes. However, a lack of information about YAP1(N) expression in that study and another report that wild-type YAP1 expression was diminished in type B3 thymoma and thymic carcinoma warrants further studies for YAP1 expression in TETs. Thus, we immunohistochemically examined YAP1(N) and YAP1(C) staining patterns in our TET samples, including 14 cases of MT. In addition, 11 of the 14 MT cases were genetically analyzed with the formalin-fixed paraffin-embedded tissues if they harbored YAP1::MAML2 fusions. MT consistently exhibited YAP1(N)-positive and YAP(C)-negative staining, whereas type B3 thymoma and thymic carcinoma showed relatively heterogeneous staining patterns for YAP1(N) and YAP1(C) and were sometimes negative for both antibodies. Furthermore, a lower expression of YAP1 was found in type B3 compared to B2 thymomas. Among genetically analyzed 11 MT cases, 6 cases showed YAP1::MAML2 fusions, whereas the analysis failed in 5 very old cases due to poor RNA quality. These results indicate that IHC of both YAP1(N) and YAP1(C) is recommended to obtain staining patterns almost unique to MT. The biological significance of YAP1 in high-grade TETs warrants further investigation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Thymoma is closely associated with myasthenia gravis (MG). However, due to the heterogeneity of thymoma and the intricate pathogenesis of MG, it remains unclear why some patients with thymoma develop MG and others do not. In this study, we conducted a comparative phenotype analysis of thymocytes in type B thymomas in patients with MG (MG (+) thymomas) and without MG (MG (-) thymomas) via fluorescence-activated cell sorting (FACS). Our results show that the developmental stages defined by the expression of CD3, CD4, and CD8 were largely maintained in both MG (+) and MG (-) thymomas, with CD4 + CD8 + cells constituting the majority of thymocytes in type B thymoma, and no significant difference between this cell population was observed in MG (+) and MG (-) thymomas.We discovered that CD4 + CD8 + thymocytes in MG (+) thymomas expressed low levels of αβ TCR and high levels of IL-7 receptor α (IL-7Rα), whereas in MG (-) thymomas, CD4 + CD8 + thymocytes exhibited the opposite pattern of αβ TCR and IL-7Rα expression. These results suggest that the positive and negative selection processes of CD4 + CD8 + thymocytes might differ between MG (+) thymomas and MG (-) thymomas. The expression of the Helios transcription factor is induced during negative selection and marks a group of T cells that have undergone negative selection and are likely to be deleted due to strong TCR binding with self-peptides/MHC ligands. We observed that the percentage of Helios-positive CD4SP T cells was greater in MG (-) than in MG (+) thymomas. Thus, the differentially regulated selection process of CD4 + CD8 + thymocytes, which involves TCR and IL-7/IL-7Rα signaling, is associated with the presence of MG in type B thymomas.

Background: Early diagnosis remains difficult because the early symptoms of thymoma are atypical., Objectives: This study aimed to analyze the changes of serum proteins in the early stage of thymoma (stage I/II) by proteomics method and to screen and validate candidate biomarkers., Methods: Proteins were extracted from 8 sera patients with stage I/II thymoma and 9 healthy controls. The levels of serum proteins were detected by data-independent acquisition (DIA) quantitative proteomics techniques, and the differential proteins were identified. The proteomic results were verified by enzyme-linked immunosorbent assay. Additionally, differentially expressed proteins were analyzed using receiver operating characteristic curves (ROC)., Results: There were 80 differentially expressed proteins between the patients with thymoma and the healthy control group, among which 39 were up-regulated and 41 were down-regulated. Differential protein enrichment is involved in environmental information processing, signaling molecules and interactions, and in the body system and the immune system. The analysis of receptor working characteristic curves showed that the areas under the curve of CORO1A, SAA1 and LTA4H were all larger than 0.8, indicating that these proteins had good diagnostic value., Conclusion: CORO1A, SAA1 and LTA4H may be new biomarkers for early screening of thymoma., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Aims: Metaplastic thymoma is a rare thymic tumour characterized by Yes Associated Protein 1 (YAP1) and Mastermind Like Transcriptional Coactivator 2 (MAML2) gene fusions resulting from an intrachromosomal inversion of chromosome 11. Immunohistochemistry with an antibody directed against the C-terminus of YAP1 has shown loss of expression in YAP1-rearranged vascular neoplasms, poromas, and porocarcinomas. This study aimed to validate an anti-YAP1 C-terminal antibody as an ancillary immunohistochemical marker for the diagnosis of metaplastic thymoma., Materials and Methods: Ten metaplastic thymomas were selected for the current study. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed to detect YAP1::MAML2 fusions. We then performed immunohistochemistry to detect YAP1 C-terminus expression in 10 metaplastic thymomas, 50 conventional thymomas (10 each of type A thymoma, type AB thymoma, type B1 thymoma, type B2 thymoma, and type B3 thymoma) and seven thymic carcinomas., Results: All 10 cases showed narrow split signals with a distance of nearly two signal diameters and sometimes had false-negative results in YAP1 and MAML2 break-apart FISH (BA-FISH). Abnormal colocalized signals of the YAP1::MAML2 fusion were observed in all 10 cases using fusion FISH (F-FISH) assays. Eight of 10 cases with adequate nucleic acids were successfully sequenced and all showed YAP1::MAML2 fusions; in two cases the fusions were detected by both DNA and RNA sequencing and in six cases by RNA sequencing only. YAP1::MAML2 fusion transcripts were identified in four cases by RT-PCR. Metaplastic thymoma showed loss of YAP1 C-terminus expression in all 10 (100%) cases. All other thymic neoplasms showed retained YAP1 C-terminus expression., Conclusion: YAP1 C-terminus immunohistochemistry is a highly sensitive and specific ancillary marker that distinguishes metaplastic thymoma from its mimics. BA-FISH assays could not effectively detect YAP1::MAML2 fusions due to the proximity of the two genes. Loss of YAP1 C-terminus expression is a reliable surrogate for the detection of YAP1::MAML2 fusions in metaplastic thymoma., (© 2023 John Wiley & Sons Ltd.)

Aims: The reliable classification of type A versus type B3 thymomas has prognostic and therapeutic relevance, but can be problematic due to considerably overlapping morphology. No immunohistochemical markers aiding in this distinction have been published so far., Methods and Results: We identified and quantified numerous differentially expressed proteins using an unbiased proteomic screen by mass spectrometry in pooled protein lysates from three type A and three type B3 thymomas. From these, candidates were validated in a larger series of paraffin-embedded type A and B3 thymomas. We identified argininosuccinate synthetase 1 (ASS1) and special AT-rich sequence binding protein 1 (SATB1) as highly discriminatory between 34 type A and 20 type B3 thymomas (94% sensitivity, 98% specificity and 96% accuracy). Although not the focus of this study, the same markers also proved helpful in the diagnosis of type AB (n = 14), B1 (n = 4) and B2 thymomas (n = 10)., Conclusions: Mutually exclusive epithelial expression of ASS1 in 100% of type B3 thymomas and ectopic nuclear expression of SATB1 in 92% of type A thymomas support the distinction between type A and type B3 thymomas with 94% sensitivity, 98% specificity and 96% accuracy., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Intrathyroid thymic carcinoma (ITC) is a rare malignant tumour. We present nine cases of ITC that were analysed by immunohistochemical staining, of which five were analysed using whole exome sequencing (WES). These cases included six women and three men with an age range of 31-66 years. The average postoperative follow-up term was 37.8 months (range, 7-95 months), and all patients survived well except for one case with lung metastasis. Microscopically, ITC showed solid islands of tumour cells separated by fibrous connective tissue containing lymphocytes and other inflammatory cells. Tumour cells strongly expressed Ckpan (AE1/AE3), P63, and CD117. And all cases but one were positive for CD5. The median value of Ki-67 was 32% (range 10-60%). We observed partial positivity of Syn and CgA in only one case. ITC shares morphological and immunohistochemical similarities with thymic squamous cell carcinoma. In situ hybridization of EBER showed negative results. All cases were microsatellite stable, and the tumour mutational burden of the 5 cases was all < 1 mutations/Mb. WES showed higher mutation rates for N4BP1 (2/5), and many genetic alterations were related to the NF-kB signalling pathway, which is crucial for insight into the molecular mechanisms of the occurrence and development of ITC., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Dendritic cells (DCs) play critical roles in the pathogenesis of myasthenia gravis (MG), and a series of DC-based experimental strategies for MG have recently been developed. However, the definite roles of different DC subsets in the mechanism of MG have scarcely been covered by previous studies. The present study aimed to investigate the levels of three main DC subsets, plasmacytoid DCs (pDCs) (CD303 positive) and two distinct subsets of conventional DCs (cDCs), namely CD1c+ cDCs and CD141+ cDCs, in MG patients and analyze related clinical features., Methods: From January 2016 to December 2020, 160 newly diagnosed MG patients and matched healthy controls (n = 160) were included in the study, and their clinical data were collected. The blood samples from MG patients before treatment and controls were collected for flow cytometry analysis. A total of 14 MG thymoma, 24 control thymoma, and 3 thymic cysts were used to immunostain the DC subsets., Results: The flow cytometry analysis showed a significantly higher frequency of circulating pDCs, CD1c+ cDCs, and CD141+ cDCs in MG patients than in healthy controls (p &lt; 0.001 for all). Patients with early-onset MG (&lt;50 years old) had a lower frequency of circulating pDCs but a higher frequency of circulating CD1c+ cDCs than those with late-onset MG (≥50 years old) (p = 0.014 and p = 0.025, respectively). The frequency of circulating pDCs was positively associated with the clinical severity of late-onset MG patients (r = 0.613, p &lt; 0.001). 64.3% (9/14) of MG thymoma is of type B2 under the World Health Organization classification, which is higher than that in control thymoma (33.3%, 8/24) (p = 0.019). For type B2 thymoma, there were significantly more pDCs but fewer CD1c+ cDCs in MG thymoma than in the controls., Conclusion: The distribution of aberrant pDCs, CD1c+ cDCs, and CD141+ cDCs in MG patients displayed age- and thymoma-related differences, which may contribute to the impaired immune tolerance and lead to the onset of MG., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Background: Regulatory B cells (Bregs) are a subset of B cells that secrete interleukin 10 (IL-10) and play a vital role in suppressing the immune response. The aim of this study was to evaluate the proportion of Bregs in patients with thymoma., Methods: The proportions of subgroups of Bregs in 23 patients with thymoma and 15 healthy controls were detected by flow cytometry. The serum IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α levels of the subjects were measured using a cytometric bead array (CBA)., Results: The proportions of circulating IL-10 + B cells, IL-10 + CD24 hi CD38 hi Bregs, and IL-10 + CD24 hi CD27 + Bregs and the serum IL-10 level were significantly higher in patients with thymoma than in the control group and were negatively correlated with the Karnofsky Performance Scale (KPS) score. The serum levels of cytokines IL-2, IL-6, IFN-γ, and TNF-α were higher and serum IL-17A level was lower in patients with thymoma. Patients with advanced-stage thymoma exhibited significantly higher proportions of IL-10-producing Bregs and a higher serum IL-10 level. After tumour resection, the frequency of circulating IL-10 + CD24 hi CD38 hi Bregs and the serum IL-10 level were significantly decreased in patients with thymoma. The serum IL-10 levels exhibited the best accuracy in assessing the risk of thymoma occurrence in this study., Conclusions: The expression of IL-10 produced by Bregs is increased in patients with thymoma, particularly those with advanced-stage disease, which may suggest that Bregs are involved in the pathogenesis and progression of thymoma.

Background: Thymic epithelial tumours (TETs) are rare tumours comprised of thymomas and thymic carcinoma. Novel therapies are needed, especially in thymic carcinoma where the 5-year survival rate hovers at 30%. Mesothelin (MSLN), a surface glycoprotein that is cleaved to produce mature MSLN (mMSLN) and megakaryocyte potentiating factor (MPF), is expressed in limited tissues. However, its expression is present in various cancers, including thymic carcinoma, where it is expressed in 79% of cases., Methods: We utilised flow cytometry, in vitro cytotoxicity assays, and an in vivo xenograft model in order to demonstrate the ability of the MSLN targeting antibody-drug conjugate (ADC) anetumab ravtansine (ARav) in inhibiting the growth of thymic carcinoma., Results: Thymoma and thymic carcinoma cell lines express MSLN, and anetumab, the antibody moiety of ARav, was capable of binding MSLN expressing thymic carcinoma cells and internalising. ARav was effective at inhibiting the growth of thymic carcinoma cells stably transfected with mMSLN in vitro. In vivo, 15 mg/kg ARav inhibited T1889 xenograft tumour growth, while combining 7.5 mg/kg ARav with 4 mg/kg cisplatin yielded an additive effect on inhibiting tumour growth., Conclusions: These data demonstrate that anetumab ravtansine inhibits the growth of MSLN positive thymic carcinoma cells in vitro and in vivo., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Thymic epithelial tumors (TETs) have been characterized at the molecular level through bioptic sections and cell lines. Despite these advances, there is a need for a more thorough characterization of the thymic stroma in thymoma, particularly because of the diversity of cell types that populate the tumor and the absence of a healthy thymic counterpart. Recent work on healthy pediatric thymi - both in vitro and at the single-cell level - now sets the stage for new studies on their neoplastic counterparts. Furthermore, general transcription factor IIi (GTF2I), a thymoma-specific oncogene, as well as some of its SNPs, are increasingly associated with autoimmune disease, a significant feature of thymomas. We summarize recent discoveries in the field and discuss the development of new targeted therapies., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The expression of immunohistochemical markers has been extensively investigated in thymomas to assist in the differential diagnosis. We have studied six select markers to determine their utility in the evaluation of these tumors. A series of 126 thymomas including 33 type A, 27 type AB, 20 type B1, 22 type B2, and 24 type B3, were examined utilizing a tissue microarray (TMA) technique with antibodies to e-cadherin, β-catenin, PAX8, bcl-2, EMA, and MIB-1. Keratin AE1/AE3 and p63 were used for quality control. A significant finding was strong and consistent positivity for bcl-2 in type A (90%) and type AB (88.8%) thymoma, while 100% of B1, B2, and B3 were negative. The distribution of e-cadherin and β-catenin was not useful for differential diagnosis. E-cadherin and β-catenin were expressed in a high proportion of all the tumors (92-100%), except for B2 thymoma which showed only 45% expression. A significant increase in the expression of the MIB-1 proliferation marker (mean: 12.8% nuclear positivity) was also observed in B3 thymoma compared with the other histologic types. Statistical significance was confirmed using Kruskal's non-parameterized test for distribution. EMA was generally negative except for spindle cells in the fibrous septa in types A and AB thymoma. PAX8 showed less consistent nuclear staining than p63 and was only widely expressed in 55.7% of cases. Bcl-2 may serve as a useful marker to separate spindle cell thymomas (Type A and AB) from the other types, and the MIB1 proliferation index may be of use to differentiate type B2 from type B3 thymoma., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Micronodular thymic carcinoma with lymphoid hyperplasia (MNTCLH) is a rare form of thymic carcinoma. We present the experience of RYTHMIC, the French national network devoted to the treatment of thymic epithelial tumors through multidisciplinary tumor boards with a review of all tumors by pathologists for classification and staging. Six cases of MNTCLH were diagnosed during a review of 1007 thymic epithelial tumors. Histologically, epithelial cells with atypia and mitoses formed micronodules that were surrounded by an abundant lymphoid background with follicles. There was neither obvious fibro-inflammatory stroma nor necrosis. Spindle cells areas were common. Initial diagnosis was micronodular thymoma in two cases, cellular atypia being overlooked, eclipsed by the micronodular pattern. Immunohistochemistry with a panel of five antibodies showed that cytokeratins (AE1-AE3) and p63-positive epithelial cells also expressed CD5 and that there was no TdT-positive cells within the tumors. CD20 highlighted the lymphoid hyperplasia. Additionally epithelial cells also expressed CD117 and diffusely Glut 1. Twenty-seven micronodular thymomas with lymphoid stroma diagnosed during the same period did not show the CD5 and CD117 positivities seen in MNTCLH and contained TdT-positive lymphocytes. Three of the 6 patients with MNTCLH had adjuvant radiotherapy. Three patients with follow-up information were alive without recurrence at 38, 51, and 95 months. Our study shows that immunohistochemistry, such as that used in the RYTHMIC network with a small panel of antibodies, may easily help to confirm the correct diagnosis of MNTCLH, a rare and low-aggressive form of thymic carcinoma, and avoid the misdiagnosis of micronodular thymoma., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Objective: To understand the characteristics of DNA methyltransferase 3a (DNMT3a) in thymoma associated Myasthenia Gravis reveal its transcriptional regulator network as while as analyze the effect of DNMT3a on Rel/ nuclear factor-kappaB family (RelA/RelB) and its downstream autoimmune regulatory factor (Aire)., Methods: Tissues of 30 patients with thymoma, with or without myasthenia gravis (MG), were collected and the DNMT3a protein expression were evaluated through immunohistochemistry. We performed mRNA expression profiling microarray detection and analysis, and integrated the analysis by constructing protein-protein interaction networks and the integration with other database. We identified molecular difference between low and high DNMT3a in the thymoma by heatmap. We also performed PCR validation in thymoma tissues. The DNMT3a-shRNA plasmid was transfected into TEC cells, and these cells were treated with 5-aza-2-deoxycytidine, a blocker of DNMT3a. After the down-regulation of DNMT3a in TEC cells, the transcript and protein levels of RelA, RelB, Aire, and CHRNA3 were evaluated by western blotting. In addition, changes in gene expression profiles were screened through microarray technology. We performed differential gene analysis in the thymoma cohort by heatmap with R (v.4.3.0) software., Results: In 30 matched tissue specimens, the expression of DNMT3a protein in thymoma with MG was lower than that in thymoma. Through mRNA expression profiling analysis, we constructed a co-expression network of DNMT3a and found direct interaction between IKZF1 and DNMT3a, and this co-expression relationship was overlappted with Cistrome DB database. We found up-regulation of 149 mRNAs and repression of 177 mRNAs in thymoma with MG compared with thymoma. Gene ontology and pathway analysis show the involvement of a multitude of genes in the mis-regulation of MG-related pathways. RNA interference significantly reduced the level of mRNA of DNMT3a, which proved that plasmid DNMT3a was effective. In comparison to the control group, the levels of DNMT3a, Aire, and CHRNA3 mRNA and protein in TEC cells transfected with DNMT3a-shRNA interference plasmid were significantly decreased, while the expression level of RelA and RelA/RelB was significantly increased., Conclusions: Our study reveals the DNMT3a-NF-κB pathway has a major effect on MG, and can be used as a marker for diagnosis as well as a target for MG treatment., (Copyright © 2021. Published by Elsevier B.V.)

Anti-PD-1 therapies can activate tumor-specific T cells to destroy tumors. However, whether and how T cells with different antigen specificity and affinity are differentially regulated by PD-1 remain vaguely understood. Upon antigen stimulation, a variety of genes is induced in T cells. Recently, we found that T cell receptor (TCR) signal strength required for the induction of genes varies across different genes and PD-1 preferentially inhibits the induction of genes that require stronger TCR signal. As each T cell has its own response characteristics, inducibility of genes likely differs across different T cells. Accordingly, the inhibitory effects of PD-1 are also expected to differ across different T cells. In the current study, we investigated whether and how factors that modulate T cell responsiveness to antigenic stimuli influence PD-1 function. By analyzing TCRs with different affinities to peptide-MHC complexes (pMHC) and pMHCs with different affinities to TCR, we demonstrated that PD-1 inhibits the expression of TCR-inducible genes efficiently when TCR:pMHC affinity is low. In contrast, affinities of peptides to MHC and MHC expression levels did not affect PD-1 sensitivity of TCR-inducible genes although they markedly altered the dose responsiveness of T cells by changing the efficiency of pMHC formation, suggesting that the strength of individual TCR signal is the key determinant of PD-1 sensitivity. Accordingly, we observed a preferential expansion of T cells with low-affinity to tumor-antigen in PD-1-deficient mice upon inoculation of tumor cells. These results demonstrate that PD-1 imposes qualitative control of T cell responses by preferentially suppressing low-affinity T cells., Competing Interests: The authors declare no competing interest.

BACKGROUND Thymoma is the most common tumor of the anterior mediastinum, and can be caused by infrequent malignancies arising from the epithelial cells of the thymus. Unfortunately, blood-based diagnostic markers are not currently available. High-throughput sequencing technologies, such as RNA-seq with next-generation sequencing, have facilitated the detection and characterization of both coding and non-coding RNAs (ncRNAs), which play significant roles in genomic regulation, transcriptional and post-transcriptional regulation, and imprinting and epigenetic modification. The knowledge about fusion genes and ncRNAs in thymomas is scarce. MATERIAL AND METHODS For this study, we gathered large-scale RNA-seq data belonging to samples from 25 thymomas and 25 healthy thymus specimens and analyzed them to identify fusion genes, lncRNAs, and miRNAs. RESULTS We found 21 fusion genes, including KMT2A-MAML2, HADHB-REEP1, COQ3-CGA, MCM4-SNTB1, and IFT140-ACTN4, as the most frequent and significant in thymomas. We also detected 65 differentially-expressed lncRNAs in thymomas, including AFAP1-AS1, LINC00324, ADAMTS9-AS1, VLDLR-AS1, LINC00968, and NEAT1, that have been validated with the TCGA database. Moreover, we identified 1695 miRNAs from small RNA-seq data that were overexpressed in thymomas. Our network analysis of the lncRNA-mRNA-miRNA regulation axes identified a cluster of miRNAs upregulated in thymomas, that can trigger the expression of target protein-coding genes, and lead to the disruption of several biological pathways, including the PI3K-Akt signaling pathway, FoxO signaling pathway, and HIF-1 signaling pathway. CONCLUSIONS Our results show that overexpression of this miRNA cluster activates PI3K-Akt, FoxO, HIF-1, and Rap-1 signaling pathways, suggesting pathway inhibitors may be therapeutic candidates against thymoma.

Cancer testis antigens (CTAs) are detected in cancer cells but not in healthy normal tissues, with the exception of gametogenic tissues. However, to our knowledge, expression of the antigens in thymic epithelial tumors has not been examined yet. We examined the immunohistochemical expression of five CTAs (MAGE-A, NY-ESO-1, MAGE-C1, SAGE and GAGE7) in 192 cases of thymic epithelial tumor. The CTAs were variably expressed in the thymic epithelial tumors. Type B component of type AB thymomas, type B1/B2/B3 thymomas, and thymic carcinomas showed a generally positive correlation between the malignancy grades and positive expression rates in four CTAs other than MAGE-C1. In thymic squamous cell carcinomas (SqCCs), four antigens except for MAGE-C1 showed high expression rates ranging from 23.1% to 43.6%. In the prognostic analysis, a positive expression of SAGE (P = 0.0485) and GAGE7 (P = 0.0289) were associated with a shorter overall survival in type B2/B3 thymomas, respectively. In thymic SqCC, a positive MAGE-A expression was significantly associated with an increased level of programmed death ligand in tumor-infiltrating lymphocytes (P = 0.0181). We showed (i) a frequent CTA expression, (ii) a general correlation of CTA expression with tumor malignancy grades and (iii) a prognostic impact in some of the CTAs., (© 2021 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Abstract: A 68-year-old man with a history of prostate cancer post-primary treatment presented with rising prostate-specific antigen levels and was referred for 18F-fluciclovine PET/MRI to localize recurrent disease. PET/MRI revealed a solitary focus of uptake in a soft tissue nodule in the anterior mediastinum, which was resected and found to be a type B2 thymoma. 18F-fluciclovine uptake is mediated by amino acid transporters, primarily alanine-serine-cysteine transporter 2 and l-type amino acid transporter 1, previously demonstrated to be expressed on thymic carcinomas. This case highlights the possibility of overexpression of amino acid transporters in thymomas as well, rarely described before., Competing Interests: Conflicts of interest and sources of funding: none related., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

This study aimed to investigate the association between the volume-dependent parameters in 18 F-fluorodeoxyglucose-positron emission tomography ( 18 F-FDG PET/CT) and a recurrence of thymic carcinoma. A retrospective chart review was performed based on our multi-institutional database to identify patients undergoing PET prior to resection of thymic carcinoma or neuroendocrine carcinoma between 1991 and 2018. The PET parameters (metabolic tumor volume and total lesion glycolysis) were evaluated retrospectively. The relevant factors were extracted and a survival analysis was performed using the Kaplan-Meier method. Sixteen patients were thus deemed to be eligible for analysis. The median follow-up period following resection was 2.65 years (range: 0.96-0.68 years). The recurrence-free survival was significantly longer in patients with a metabolic tumor volume <  = 22.755 cm 3 and with total lesion glycolysis <  = 105.4006 g/mL (p = 0.001 and 0.001, respectively, by a log-rank test). The metabolic tumor volume and total lesion glycolysis may, therefore, be predictive of the postoperative recurrence of thymic carcinoma.

Thymomas exhibit a unique genomic landscape, comprising the lowest on average total mutational burden among adult human cancers; a unique point mutation in the GTF2I gene in WHO type A and AB thymomas (and rarely others); almost unique KMT2A-MAML2 translocations in rare WHO type B2 and B3 thymomas; a unique YAP1-MAML2 translocation in almost all metaplastic thymomas; and unique miRNA profiles in relation to GTF2I mutational status and WHO histotypes. While most thymomas can be diagnosed solely on the basis of morphological features, mutational analyses can solve challenging differential diagnostic problems. No molecular biomarkers have been identified that predict the response of unresectable thymomas to chemotherapy or agents with known molecular targets. Despite the common and strong expression of PDL1 in thymomas, immune checkpoint inhibitors are rarely applicable due to the poor predictability of common, life-threatening autoimmune side effects that are related to the unrivaled propensity of thymomas towards autoimmunity.

The present study reported a rare case with thymic carcinoid as the first manifestation of multiple endocrine neoplasia type 1 (MEN1) syndrome, which presented with gene mutations of the MEN1 and glucokinase regulatory protein (GCKR). In this report, a 40-year-old male was diagnosed as MEN1 syndrome with thymic carcinoid, pancreatic cancer, hyperparathyroidism, and insulinoma with intrahepatic metastasis. Genetic testing showed the mutations of the MEN1 (c.378 G>A, p. Trp126*) in the patient, his children and two sisters, and GCKR (c.151C>T, p. Arg51*) gene in the patient and his children. The pathological examination showed that neuroendocrine tumor (NET) of the pancreas was characterized as 6 mitoses per 10 high-power fields (HPF), infiltration of adipose tissue, no intravascular tumor thrombus and nerve infiltration. In addition, NET of the liver was characterized as 4 mitoses per 10 HPF, no intravascular tumor thrombus and nerve infiltration. Immunohistochemical staining showed Ckpan (+), Syn (+), CgA (+), CD 56 (+), PGP 9.5 (+), and Ki67-positive cells (8-10%) in NET. Therefore, we suggested that genetic testing in family members of MEN1 patient, which may be helpful for early diagnosis., (© 2020 by the Association of Clinical Scientists, Inc.)

Background: It is popularly believed that myasthenia gravis (MG) patients show acetylcholine receptor antibody (AChRAb) production associated with the thymus (germinal centers, approximately 80%). It has been suggested that thymectomy can remove the area of autoantibody production. This study aimed to determine whether the solid volume of the thymus calculated using three-dimensional (3D) imaging could be used to predict the efficacy of thymectomy. Additionally, the study assessed the relationships of the solid volume with germinal centers, change in the serum AChRAb level, postoperative MG improvement, and prednisolone (PSL) dose reduction extent., Methods: This retrospective study included 12 consecutive non-thymomatous MG patients (9 female and 3 male patients), who underwent extended thymectomy at our institution over the last 10 years. The mean patient age was 43.3 ± 14.2 years (range, 12-59 years). The study assessed the number of germinal centers per unit area, change in the serum AChRAb level, postoperative MG improvement, PSL dose reduction extent, and solid volume of the thymus., Results: The number of germinal centers per unit area was significantly correlated with the solid volume of the thymus. The PSL dose reduction extent tended to be correlated with the solid volume., Conclusions: Our findings suggest that the solid volume of the thymus can possibly predict steroid dose reduction. Additionally, the solid volume of the thymus in 3D images is the most important indicator for predicting the efficacy of extended thymectomy., Competing Interests: The authors have declared that no competing interests exist.

Previous studies have shown that cancer susceptibility candidate 5 (CASC5) plays important roles in several types of cancer. But its expression and clinical significance in human pan-cancer remain largely unclear. In the present study, we comprehensively analysed the expression profile and prognostic values of CASC5 in pan-cancer across 33 cancer types based on the online TCGA analysis databases. CASC5 was found to be abnormally expressed in 16 types of cancer. In addition, dysregulated expression of CASC5 was closely associated with patient overall survival (OS) in kidney renal papillary cell carcinoma (KIRP), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD) and thymoma (THYM). By comparative analysis, we found that CASC5 was significantly up-regulated in LUAD and predicted poor patient OS. High CASC5 expression was closely correlated with tumour advanced stages of patients with LUAD. Through GSEA based on the KEGG database, CASC5 was found to be closely related to DNA replication and microRNA regulation in LUAD. Functionally, knockdown of CASC5 could inhibit cell proliferation of LUAD cells in vitro, rather than affecting cell migration and invasion. Mechanistically, CASC5 promoted proliferation of LUAD cells by targeting miR-139-5p. Collectively, our findings reveal that CASC5 is a novel oncogenic gene in LUAD and may be a potential clinical target and (or) biomarker for this human malignancy. SIGNIFICANCE OF THE STUDY: In this study, we for the first time comprehensively analysed the transcriptional level and prognostic significance of CASC5 in human pan-cancer across 33 cancer types using online TCGA databases. Our study indicates that CASC5 is aberrantly expressed in many tumours and is closely related to the patient overall survival of several tumour types. Our findings reveal that CASC5 is a novel oncogene in LUAD based on bioinformatic analysis and functional experiments. Mechanistically, CASC5 promoted LUAD proliferation by targeting miR-139-5p. Results of this study suggest that CASC5 is a potential clinical target and (or) biomarker for LUAD., (© 2020 John Wiley & Sons Ltd.)

Background: KIT proto-oncogene ligand (KITLG) is a pleiotropic factor which is found in diverse cancers and is involved in cell proliferation, differentiation, and survival. However, the value of KITLG in thymoma remains unclear., Methods: A total of 121 thymoma samples from The Cancer Genome Atlas Thymoma (TCGA-THYM) dataset were used to analyze KITLG related genome-wide expression profiles, and microRNA profiles and methylation alterations and a GEO dataset-GSE29695, including 37 samples was used as verification. For cell-based studies, specific small interfering RNA targeting KITLG or a KITLG overexpression vector were used to clarify the changes of the MAPK pathway in an AB thymoma cell line Thy0517., Results: Both datasets showed that high expression of KITLG was significantly associated with type A and AB thymoma. Through multiomic analysis of the TCGA-THYM, it was found that with the high expression of KITLG, there were 220 upregulated and 72 downregulated genes at the mRNA level, 79 positive and 78 negative miRNAs, 28 hypermethylation and 163 hypomethylation regions. In the thymoma cell line Thy0517, it was found that the expression of GRB2 and the phosphorylation levels of BRAF, MEK1/2, and ERK1/2 in the MAPK pathway were positively correlated with the change in KITLG., Conclusions: High expression of KITLG is a new hallmark of WHO type A and AB thymomas in which it might play a critical role through the activation of the MAPK signaling pathway. Additionally, it is hoped that KITLG will become a potential target for the diagnosis of type A and AB thymoma through further research in the future., Key Points: SIGNIFICANT FINDINGS OF THE STUDY: KIT proto-oncogene ligand (KITLG) is a new hallmark of type A and AB thymomas which induce a series of aberrant alteration of mRNA, miRNA and DNA methylation. The expression of KITLG is significantly higher in type A and AB than other subtypes of thymoma., What This Study Adds: KITLG activated the MAPK signaling pathway to promote type A and AB thymoma which might be a potential diagnostic biomarker or target., (© 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Imbalance of T helper 17 (Th17)/regulatory T (Treg) cells is involved in the pathogenesis of myasthenia gravis with thymoma (MG-T). Long non-coding RNAs (lncRNAs) are implicated in the regulation of Th17/Treg balance. This study was designed to explore the role of XLOC&#95;003810, a novel lncRNA, in regulating the Th17/Treg balance in MG-T. The thymic CD4 + T cells were isolated from control subjects and MG-T patients. The Th17/Treg balance was evaluated by determining proportions of Th17 and Treg cells and expression of Th17- and Treg- associated molecules. Lentivirus-mediated silencing and overexpression of XLOC&#95;003810 in CD4 + T cells were performed. The results showed that XLOC&#95;003810 expression was higher in MG-T thymic CD4 + T cells than that in the control group. Furthermore, the ratio of Th17/Treg cells, proportion of Th17 cells and levels of Th17-associated molecules were significantly increased, whereas the proportion of Treg cells and levels of Treg-associated molecules were decreased in MG-T thymic CD4 + T cells. Importantly, the Th17/Treg imbalance in MG-T thymic CD4 + T cells was aggravated by XLOC&#95;003810 overexpression, whereas it was attenuated by XLOC&#95;003810 silencing. Collectively, XLOC&#95;003810 modulates thymic Th17/Treg balance in MG-T patients, providing the scientific basis for the clinical targeted therapy of MG-T., (© 2020 John Wiley & Sons Australia, Ltd.)

Although clinical responses with CD19-targeting chimeric antigen receptor (CAR) T-cell treatment have been observed in patients with certain hematologic malignancies, high rates of disease relapse highlight the necessity to understand and improve mechanisms of CAR T-cell failure. Because T-cell dysfunction is thought to contribute to CAR T-cell treatment failure, understanding what mechanisms drive T cells into this dysfunctional state may aid optimal design of efficacious CAR T cells. Dysfunctional CAR T cells have been characterized as having upregulated inhibitory receptors and decreased cytolytic capabilities. Previous studies have identified a role for sustained CAR CD3ζ signaling in CAR T-cell dysfunction. Here, we demonstrate a mechanism that drives dysfunction in CAR T cells through excessive costimulation. Fully activated CD19-targeted CAR T cells were rendered dysfunctional upon stimulation with both endogenous CD28 stimulation and CAR-mediated CD28 costimulation. Costimulation-driven dysfunction of CAR T cells was demonstrated in a syngeneic immunocompetent mouse model, in which CAR T cells were activated with signals 1 (CD3ζ), 2 (CD28), and 3 (IL12). Thus, we show that CAR T-cell dysfunction can be driven through excessive CD28 and 4-1BB costimulation. See related article by Drakes et al., p. 743 ., (©2020 American Association for Cancer Research.)

Patient stratification and individualized therapeutic strategies rely on the established knowledge of genotype-specific molecular and cellular alterations of biological and therapeutic significance. Whilst almost all approved drugs have been developed based on the Reference Sequence protein database (RefSeq), the latest genome sequencing studies establish the substantial prevalence of non-synonymous genetic mutations in the general population, including stop-insertion and frame shift mutations within the coding regions of membrane proteins. While the availability of individual genotypes are becoming increasingly common, the biological and clinical interpretations of mutations among individual genomes is largely lagging behind. Lately, transmembrane proteins of haematopoietic (myeloid and lymphoid) derived immune cells have attracted much attention as important targets for cancer immunotherapies. As such, the signalling properties of haematological transmembrane receptors rely on the membrane-proximal phosphotyrosine based sequence motifs (TBSMs) such as ITAM (immunoreceptor tyrosine-based activation motif), ITIM (immunoreceptor tyrosine-based inhibition motif) and signal transducer and activator of transcription 3 (STAT3)-recruiting YxxQ motifs. However, mutations that alter the coding regions of transmembrane proteins, resulting in either insertion or deletion of crucial signal modulating TBSMs, remains unknown. To conveniently identify individual cell line-specific or patient-specific membrane protein altering mutations, we present the Transmembrane Protein Sequence Variant Identifier (TraPS-VarI). TraPS-VarI is an annotation tool for accurate mapping of the effect of an individual's mutation in the transmembrane protein sequence, and to identify the prevalence of TBSMs. TraPS-VarI is a biologist and clinician-friendly algorithm with a web interface and an associated database browser (https://www.traps-vari.org/).

Thymomas are associated with a very high risk of developing Myasthenia Gravis (MG). Our objectives were to identify histological and biological parameters to allow early diagnosis of thymoma patients susceptible to developing MG. We conducted a detailed retrospective analysis from a patient database, searching for differences between patients with thymoma-associated MG (MGT, n = 409) and thymoma without MG (TOMA, n = 111) in comparison with nonthymomatous MG patients (MG, n = 1246). We also performed multiplex and single molecule arrays to measure the serum levels of cytokines in these groups of patients and controls (n = 14-22). We identified a set of parameters associated with MG development in thymoma patients: 1) detection of anti-acetylcholine receptor (AChR) antibodies, 2) development of B1 or B2 thymoma subtypes, 3) presence of ectopic thymic germinal centers (GCs), 4) local invasiveness of thymoma, and 5) being a woman under 50 years old. Among these parameters, 58.8% of MGT patients displayed GCs with a positive correlation between the number of GCs and anti-AChR titers. By immunohistochemistry, we found thymic GCs in the adjacent tissues of thymomas encircled by high endothelial venules (HEVs) that could favor peripheral cell recruitment. We also clearly associated MG symptoms with higher IFN-γ, IL-1β and sCD40L serum levels, specifically in MGT patients compared to TOMA patients. Altogether, these analyses allowed the clear identification of histological, in particular the presence of GCs, and biological parameters that would facilitate the evaluation of the probability of the MG outcome postoperatively in thymoma patients., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Various immune cells are recruited in the tumor microenvironment. It is well established that cellular immune responses, such as cytotoxic or suppressive activities, play an important role in regulating tumor growth and metastasis. However, the contribution of humoral immune responses against tumors is poorly understood. Fc receptors constitute critical elements for the up- or downregulation of immune responses through immune complexes. Here, we examined the potential role of the inhibitory Fc receptor, Fcγ receptor IIB (FcγRIIB), in tumor immunity using a mouse model. Our findings indicated that tumor-specific antibodies are induced in tumor-bearing mice and control tumor immunity. FcγRIIB deletion significantly improved both cellular and humoral immunity against tumors and delayed tumor growth. These findings indicated that spontaneous antibodies against tumors create a suppressive tumor microenvironment through FcγRIIB signaling, thus suggesting an attractive therapeutic target for cancer immunotherapy.

Intrathyroidal thymic carcinoma is a malignant epithelial tumor of the thyroid gland with thymic epithelial differentiation; it is the malignant counterpart of ectopic thymoma of the thyroid gland and histologically resembles thymic carcinoma. Given its favorable prognostic course, preoperative cytological diagnosis is important to distinguish this neoplasm from other more aggressive thyroid neoplasms that can have similar or overlapping cytomorphological features. However, there is little information about the cytology of this disease available in the literature, even in the latest fourth edition of World Health Organization classification of tumors of endocrine organs. Here, we report a case of intrathyroidal thymic carcinoma exhibiting neuroendocrine differentiation in a 33-year-old woman and present a review of the literature., (© 2019 Wiley Periodicals, Inc.)

Objectives: Eight cases of encapsulated and cystic atypical thymomas (World Health Organization type B3) are presented., Methods: Cases were identified after review of more than 500 thymomas. Histologic material from surgical resections was evaluated., Results: The patients are seven men and one woman aged 43 to 71 years. Seven patients presented with nonspecific symptoms while one mass was found on autopsy. The tumors were 4 to 6 cm in greatest dimension and showed prominent cystic changes with an epithelial proliferation arranged in sheets and cords. Increased mitotic activity and nuclear pleomorphism were not identified. All tumors were encapsulated. Immunohistochemical studies showed the tumor cells positive for keratin cocktail, keratin 5/6, and p63. Clinical follow-up was available in five patients. All were alive without recurrence., Conclusions: These cases represent an unusual variant of atypical thymoma that can be misdiagnosed as thymic carcinoma or multilocular thymic cyst. Awareness of this histologic growth pattern is important for accurate diagnosis., (© American Society for Clinical Pathology, 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The immune system is critical in modulating cancer progression. Pseudogenes are a special type of long non-coding RNAs that regulate different tumorigenic processes. However, the potential roles of pseudogenes in tumor-immune interaction remain largely unclear. Here, we reported that pseudogene RP11-424C20.2 and its parental gene UHRF1 were frequently up-regulated and positively correlated in liver hepatocellular carcinoma (LIHC) and thymoma (THYM), but associated with distinct clinical outcomes. We further found that RP11-424C20.2 may act as a competing endogenous RNA (ceRNA) to increase UHRF1 expression through sponging miR-378a-3p. Functional enrichment analysis showed a strong association of UHRF1 with immune-related biological processes. We also observed that UHRF1 expression significantly correlated with immune infiltration, and different types of tumor-infiltrating immune cells displayed different impacts on clinical outcomes. Furthermore, UHRF1 expression in LIHC and THYM showed an opposite correlation with biomarkers from monocyte, dendritic cell, Th1 and T cell exhaustion. Mechanism investigations revealed that RP11-424C20.2/UHRF1 axis regulated immune escape of LIHC and THYM at least partly through IFN-γ-mediated CLTA-4 and PD-L1 pathway. These findings demonstrate a disparate role of RP11-424C20.2/UHRF1 axis in LIHC and THYM via regulating immune infiltrates, and also indicate a therapeutic value for UHRF1 inhibitors in combination with anti-PD-L1/CLTA-4 blockade.

Background: Histology is a traditional way to classify subtypes of thymoma, because of low cost and convenience. Yet, due to the diverse morphology of thymoma, this method increases the complexity of histopathologic classification, and requires experienced experts to perform correct diagnosis. Therefore, in this study, we developed an alternative method by identifying protein biomarkers in order to assist clinical practitioners to make right classification of thymoma subtypes., Methods: In total, 204 differentially expressed proteins in three subtypes of thymoma, AB, B2, and B3, were identified using mass spectrometry. Pathway analysis showed that the differentially expressed proteins in the three subtypes were involved in activation-related, signaling transduction-related and complement system-related pathways. To predict the subtypes of thymoma using the identified protein signatures, a support vector machine algorithm was used. Leave-one-out cross validation methods and receiver operating characteristic analysis were used to evaluate the predictive performance., Results: The mean accuracy rates were > 80% and areas under the curve were ≧0.93 across these three subtypes. Especially, subtype B3 had the highest accuracy rate (96%) and subtype AB had the greatest area under the curve (0.99). One of the differentially expressed proteins COL17A2 was further validated using immunohistochemistry., Conclusions: In summary, we identified specific protein signatures for accurately classifying subtypes of thymoma, which could facilitate accurate diagnosis of thymoma patients.

Immunotherapies are an emerging strategy for treatment of solid tumors. Improved understanding of the mechanisms employed by cytotoxic T lymphocytes (CTL) to control tumors will aid in the development of immunotherapies. CTLs can directly kill tumor cells in a contact-dependent manner or may exert indirect effects on tumor cells via secretion of cytokines. Here, we aim to quantify the importance of these mechanisms in murine thymoma EL4/EG7 cells. We developed an agent-based model (ABM) and an ordinary differential equation model of tumor regression after adoptive transfer of a population of CTLs. Models were parameterized based on in vivo measurements of CTL infiltration and killing rates applied to EL4/EG7 tumors and OTI T cells. We quantified whether infiltrating CTLs are capable of controlling tumors through only direct, contact-dependent killing. Both models agreed that the low measured killing rate of CTLs in vivo was insufficient to cause tumor regression. In our ABM, we also simulated CTL production of the cytokine IFNγ in order to explore how an antiproliferative effect of IFNγ might aid CTLs in tumor control. In this model, IFNγ substantially reduced tumor growth compared with direct killing alone. Collectively, these data demonstrate that contact-dependent killing is insufficient for EL4 regression in vivo and highlight the potential importance of cytokine-induced antiproliferative effects in T-cell-mediated tumor control. SIGNIFICANCE: Computational modeling highlights the importance of cytokine-induced antiproliferative effects in T-cell-mediated control of tumor progression., (©2019 American Association for Cancer Research.)

Eight cases of primary thymic epithelial neoplasms corresponding to 7 thymomas and 1 thymic carcinoma with sebaceous differentiation are presented. The patients are 5 men and 3 women between the ages of 45 and 63 years (average, 54 years) who presented with nonspecific symptoms related to their mediastinal mass. All patients underwent complete surgical resection of the mediastinal mass. Grossly, all the tumors were described as round to oval measuring from 3.5 to 6.0 cm in greatest diameter. In 4 cases, the tumors were described as with infiltrative borders. Histologically, 1 tumor corresponded to thymic carcinoma characterized by irregular islands of tumors cells showing cellular atypia and mitotic activity. Six of the 7 thymomas showed mixed histologies corresponding to spindle cell, lymphocyte predominant, and mixed lymphocyte/epithelial types (World Health Organization types A, B1, and B2). One thymoma was mixed lymphocyte/epithelial (World Health Organization type B2). The areas of sebaceous differentiation characterized by clusters or strands of epithelial cells with ample clear cytoplasm were present within the lymphocytic component, whereas in the thymic carcinoma, the areas of sebaceous differentiation were identified within the epithelial component of the tumor. Follow-up information was obtained in 5 patients, showing that the patient with thymic carcinoma died 20 months after initial diagnosis, whereas the patients with thymoma whether encapsulated or minimally invasive remained alive without recurrence 12 to 24 months after initial diagnosis. The current cases represent an unusual feature that occasionally may be seen in thymomas and thymic carcinomas., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The pathogenesis of thymic epithelial tumors remains poorly elucidated. The PIK3/Akt/mTOR pathway plays a key role in various cancers; interestingly, several phase I/II studies have reported a positive effect of mTOR inhibitors in disease control in thymoma patients. A major limit for deciphering cellular and molecular events leading to the transformation of thymic epithelial cells or for testing drug candidates is the lack of reliable in vitro cell system. We analyzed protein expression and activation of key players of the Akt/ mTOR pathway namely Akt, mTOR, and P70S6K in eleven A, B and AB thymomas as well as in normal thymuses. While only Akt and phospho-Akt were expressed in normal thymuses, both Akt and mTOR were activated in thymomas. Phospho-P70S6K was expressed in all thymic tumors whatever their subtypes, and absent in normal thymus. Interestingly, we report the activation of Akt, mTOR and P70S6 proteins in primary thymic epithelial cells maintained for short period of time after their derivation from seven AB and B thymomas. Finally, we showed that rapamycin (100 nM) significantly reduced proliferation of thymoma- derived epithelial cells without inducing cell death. Our results suggest that the activation of the Akt/ mTOR pathway might participate to the cell proliferation associated with tumor growth. Ultimately, our data enhance the potential role of thymic epithelial cells derived from tissue specimens for in vitro exploration of molecular abnormalities in rare thymic tumors., Competing Interests: The authors have declared that no competing interests exist.

Background: Recent genomic and bioinformatic technological advances have made it possible to dissect the immune response to personalized neoantigens encoded by tumor-specific mutations. However, timely and efficient identification of neoantigens is still one of the major obstacles to using personalized neoantigen-based cancer immunotherapy., Methods: Two different pipelines of neoantigens identification were established in this study: (1) Clinical grade targeted sequencing was performed in patients with refractory solid tumor, and mutant peptides with high variant allele frequency and predicted high HLA-binding affinity were de novo synthesized. (2) An inventory-shared neoantigen peptide library of common solid tumors was constructed, and patients' hotspot mutations were matched to the neoantigen peptide library. The candidate neoepitopes were identified by recalling memory T-cell responses in vitro. Subsequently, neoantigen-loaded dendritic cell vaccines and neoantigen-reactive T cells were generated for personalized immunotherapy in six patients., Results: Immunogenic neo-epitopes were recognized by autologous T cells in 3 of 4 patients who utilized the de novo synthesis mode and in 6 of 13 patients who performed shared neoantigen peptide library, respectively. A metastatic thymoma patient achieved a complete and durable response beyond 29 months after treatment. Immune-related partial response was observed in another patient with metastatic pancreatic cancer. The remaining four patients achieved the prolonged stabilization of disease with a median PFS of 8.6 months., Conclusions: The current study provided feasible pipelines for neoantigen identification. Implementing these strategies to individually tailor neoantigens could facilitate the neoantigen-based translational immunotherapy research.TRIAL REGSITRATION. ChiCTR.org ChiCTR-OIC-16010092, ChiCTR-OIC-17011275, ChiCTR-OIC-17011913; ClinicalTrials.gov NCT03171220., Funding: This work was funded by grants from the National Key Research and Development Program of China (Grant No. 2017YFC1308900), the National Major Projects for "Major New Drugs Innovation and Development" (Grant No.2018ZX09301048-003), the National Natural Science Foundation of China (Grant No. 81672367, 81572329, 81572601), and the Key Research and Development Program of Jiangsu Province (No. BE2017607).

Background: Programmed death ligand 1 (PD-L1) is reportedly expressed in various malignancies and is considered a prognostic factor. We attempted to reveal the usefulness of the PD-L1 expression as a prognostic factor in patients with thymoma., Methods: Eighty-one patients with thymoma who underwent surgical resection between 2004 and 2015 were retrospectively reviewed. The PD-L1 expression was evaluated by immunohistochemistry and stratified by the proportion of positive tumor cells. Strong membranous reactivity of the PD-L1 antibody in 1% or more of tumor cells was considered "positive." The association between the PD-L1 expression and the clinicopathologic features was investigated., Results: The PD-L1 expression was positive in 22 patients (27%) and negative in 59 patients (73%). The PD-L1 positivity was significantly associated with type B2 and B3 thymoma (p < 0.001) and stage III and IV disease (p = 0.048). In addition, PD-L1 positive tumors showed a significantly higher maximum standardized uptake value than PD-L1 negative tumors (p = 0.026). The 5-year disease-free survival rate was 82% in PD-L1 positive patients and 88% in PD-L1 negative patients, showing no significant difference (p = 0.57). Furthermore, PD-L1 positivity was not an independent prognostic factor for the disease-free survival on a Cox proportional hazards analysis (p = 0.59)., Conclusions: A strong expression of PD-L1 in thymoma was significantly associated with type B2 and B3 and higher pathologic stages. In addition, PD-L1 positivity was associated with an increased maximum standardized uptake value of the tumor. However, patients with PD-L1 positive thymomas did not show a significantly worse prognosis than patients with PD-L1 negative tumors., (Copyright © 2019 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Neoplastic cells display altered biosynthetic and bioenergetic machinery to support cell survival. Therefore, cancer cells optimally utilize all available fuel resources to pump their highly upregulated metabolic pathways. While glucose is the main carbon source, transformed cells also utilize other molecules, which can be utilized in metabolic pathways, designated as alternative fuels. Acetate is one of such alternative metabolic fuels, which is mainly consumed in carbohydrate and lipid metabolism. However, studies demonstrate the contradictory effects of acetate on tumor cell survival. Moreover, the mechanisms of its antitumor actions remain poorly understood. Further, the spectrum of acetate susceptible tumor targets needs to be characterized in order to optimize the use of acetate in maneuvering tumor progression as a therapeutic strategy. As the effect of acetate on survival properties of the tumor cells of thymic origin is not worked out, in the present study the effect of acetate was investigated against tumor cells derived from a murine thymoma designated as Dalton's Lymphoma (DL). Acetate treatment of tumor cells inhibited tumor cell survival accompanied by induction of apoptotic cell death, associated with modulated expression of cell survival regulatory HIF1α, ROS, p53, Caspase 3, Bax and HSP70 along with the elevated level of cytosolic cytochrome c. Acetate treatment also modulated the expression of pH regulators MCT-1 and V-ATPase accompanied by altered pH homeostasis. Expression of MDR and lipid metabolism regulatory molecules was also inhibited in tumor cells upon acetate exposure. Further, pre-exposure of tumor cells to α-CHC (α-cyano-4-hydroxycinnamate), an inhibitor of MCT-1, partially abrogated the cytotoxic action of acetate. These findings shed a new light regarding the effect and mechanisms of the exogenous acetate on the biology of tumor cells of thymic origin., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Purpose: A thymoma is a tumor arising from the epithelium of the thymus, mostly occurring in the anterior mediastinum. The incidence of this disease is low and research progress in this field is slow. Consequently, there is an urgent need to investigate the correlation between molecular regulation and the prediction of survival and prognosis in patients with thymoma., Methods: We collected thymoma datasets from The Cancer Genome Atlas (TCGA). Differentially expressed genes (DEGs) of TCGA datasets were then determined using R software. A gene signature was obtained by screening prognostic DEGs from the TCGA datasets using univariate and multivariate Cox survival analysis. The summation of the weighted expression levels was calculated as a risk score, which could then be used to predict the survival rate and prognosis of patients with thymoma. The validity of this model was verified by the analysis of receiver operating characteristic (ROC) curves and area under the curve (AUC)., Results: In total, 297 DEGs were identified from the integrated results of TCGA datasets. A seven-gene signature, along with a regression model of prognostic risk, was obtained by Cox survival analysis. The expression levels of the seven genes were then weighted and summed to calculate the risk score for each sample. Patients were effectively divided into high- and low-risk groups using the median risk score (P < 0.05). ROC analysis showed that this Cox regression model was effective in predicting the prognosis of patients with thymus tumors (AUC = 0.983, P < 0.05)., Conclusion: For the first time, we identified an effective seven-gene signature in patients with thymic tumors. In the future, the risk and prognosis of patients can be preliminarily assessed using this model, although further testing is required to improve rigor.

Follicular cell-derived thyroid carcinomas, including thyroid squamous cell carcinomas (SCCs) and anaplastic carcinomas, are immunoreactive for paired-box gene 8 (PAX8), while non-follicular cell-derived thyroid carcinomas stain negative for the PAX8 antibody. Intrathyroid thymic carcinoma (ITTC) arising from the intrathyroidal ectopic thymus exhibits moderate-to-strong nuclear reactivity for polyclonal PAX8. This is difficult to understand given that PAX8 is not associated with embryonic thymic development. We aimed to determine the diagnostic significance of monoclonal PAX8 antibody in distinguishing ITTCs from follicular cell-derived thyroid carcinomas. Ten ITTCs, 14 poorly differentiated thyroid carcinomas (PDTCs), 14 thyroid SCCs, 7 thymic tissue specimens, 7 thymomas, and 1 thymic carcinoma were analyzed using antibodies against polyclonal and monoclonal PAX8, thyroid transcription factor-1, p63, and CD5. Four ITTCs (40.0%) stained positive for polyclonal PAX8; none stained positive for monoclonal PAX8. All PDTCs and 92.9% of SCCs were immunoreactive for both polyclonal and monoclonal PAX8. All PDTCs, 46.2% of SCCs, and none of the ITTCs were immunoreactive for thyroid transcription factor-1. Eight ITTCs (80.0%), but none of the PDTCs and SCCs, were immunoreactive for CD5. We are the first to show that ITTCs stain negative for monoclonal PAX8. Monoclonal PAX8 is a more reliable marker than polyclonal PAX8 for determining follicular cell origin. We conclude that monoclonal PAX8 is a useful marker for distinguishing ITTCs from PDTCs and SCCs. Monoclonal PAX8 negativity is additional evidence in support of ITTCs not being follicular cell-derived thyroid carcinomas, but having a thymic origin.

The antitumor activity of activated CD8+ T cells in the tumor microenvironment seems to be limited due to their being metabolically unfit. This metabolic unfitness is closely associated with T-cell exhaustion and impairment of memory formation, which are barriers to successful antitumor adoptive immunotherapy. We therefore assessed the role of glutamine metabolism in the antitumor activity of CD8 + T cells using a tumor-inoculated mouse model. The adoptive transfer of tumor-specific CD8 + T cells cultured under glutamine-restricted (dGln) conditions or CD8 + T cells treated with specific inhibitors of glutamine metabolism efficiently eliminated tumors and led to better survival of tumor-inoculated mice than with cells cultured under control (Ctrl) conditions. The decreased expression of PD-1 and increased Ki67 positivity among tumor-infiltrating CD8 + T cells cultured under dGln conditions suggested that the inhibition of glutamine metabolism prevents CD8 + T-cell exhaustion in vivo. Furthermore, the transferred CD8 + T cells cultured under dGln conditions expanded more efficiently against secondary OVA stimulation than did CD8 + T cells under Ctrl conditions. We found that the expression of a pro-survival factor and memory T cell-related transcription factors was significantly higher in CD8 + T cells cultured under dGln conditions than in those cultured under Ctrl conditions. Given these findings, our study uncovered an important role of glutamine metabolism in the antitumor activity of CD8 + T cells. The novel adoptive transfer of tumor-specific CD8 + T cells cultured in glutamine-restricted conditions may be a promising approach to improve the efficacy of cell-based adoptive immunotherapy., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Objectives: The expression of immune checkpoint ligand PD-L1 has been reported in various tumors. The expression of IDO and FOXP3 Tregs are considered to be associated with tumor-induced tolerance and poor outcome. Their prognostic role in surgically treated thymoma and thymic carcinoma, however, has not been investigated., Materials and Methods: Tissue microarray (TMA) blocks comprised of 100 surgically treated thymomas and 69 surgically treated thymic carcinomas were conducted. Tissue sections were incubated with primary antibodies against PD-L1 (clone E1L3N, 1:100), IDO (clone 10.1, 1:50), and FOXP3 (clone 236 A/E7, 1:50). Comparisons for categorical variables were performed using χ 2 test and Fisher's exact test. Survival analysis was established using Kaplan-Meier method and log-rank test. Univariate and multivariate analyses were performed using Cox regression model., Results and Conclusions: High expression of PD-L1, IDO, and FOXP3 Tregs were identified in 36 (36%), 13 (13%), and 16 (16%) thymoma patients, respectively. High expression of PD-L1, IDO, and FOXP3 Tregs was associated with higher grade of tumor histology (P <  0.001, P =  0.007, and 0.014, respectively). High expression of PD-L1 was also associated with advanced Masaoka staging (P <  0.001). In patients with thymic carcinoma, high expression of PD-L1, IDO, and FOXP3 Tregs were identified in 25 (36%), 10 (14%), and 20 (29%) patients, respectively. Complete resection, low expression of IDO, and high expression of FOXP3 Tregs were associated with better overall survival (P =  0.001, 0.004, and 0.032, respectively), and progression-free survival (P <  0.001, P =  0.026, and 0.047, respectively) in multivariate analysis. In surgically treated thymoma, high PD-L1 expression was associated with advanced Masaoka staging. High PD-L1, IDO, and FOXP3 Tregs expression was associated with high grade histology. In surgically treated thymic carcinoma, significant survival benefit was noted in patients with complete resection, low IDO expression, and high FOXP3 Tregs expression., (Copyright © 2018. Published by Elsevier B.V.)

L-type amino acid transporter 1 (LAT1) functions to transport large neutral amino acids, such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, and histidine. These amino acids are essential for cell growth and proliferation. Many studies have demonstrated LAT1 expression in various types of cancer, and its high expression level was associated with poor prognosis. However, the significance of LAT1 expression in thymic epithelial tumors is controversial. We conducted this retrospective study to investigate the LAT1 immunoreactivity in thymic epithelial tumors and its impact on prognosis. We analyzed 32 patients with thymoma and 14 patients with thymic carcinoma who underwent surgery at our institute. Immunohistochemical analysis was performed using formalin-fixed paraffin-embedded surgical tissues and an anti-LAT1 polyclonal antibody. We thus found that LAT1 immunoreactivity was undetectable in all of the thymoma specimens, regardless of the subtypes of thymoma. By contrast, LAT1 immunoreactivity was consistently detected in the cytosol of thymic carcinoma cells; namely, all 14 thymic carcinoma specimens demonstrated LAT1 immunoreactivity in the cytosol. Among these 14 thymic carcinoma specimens, four carcinoma specimens also showed LAT1 immunoreactivity in the cell membrane. Survival analysis indicated that the thymic carcinoma with the LAT1 membrane signal was associated with poor prognosis, compared with the specimens with the LAT1 cytosol signal. We therefore propose that LAT1 is expressed in the cytosol of thymic carcinoma cells, which could be a diagnostic marker of thymic carcinoma. Moreover, LAT1 expression in the cell membrane is a prognostic marker of thymic carcinoma.

Introduction: Thymic epithelial tumors (TETs) including thymoma and thymic carcinoma are rare tumors with little data available to guide treatment. Immunotherapy with checkpoint blockade has shown promising activity, but data regarding the expression patterns and prognostic implications of programmed death 1 (PD-1) and its ligand (PD-L1) in TETs have yielded conflicting results. Intratumoral heterogeneity of PD-1/L1 expression has been shown in other cancers, but has not been described in the TET literature., Methods: We performed a retrospective single-center review of 35 patients with resected TET. PD-1/L1 expression was assessed by immunohistochemistry using PD-1 clone: NAT105 and PD-L1 clone: 22C3. Tumor samples from 35 patients were evaluated including 32 patients with thymoma and 3 patients with thymic carcinoma., Results: PD-L1 expression was detected in 83% (29 of 35) tumor samples, including 100% (3 of 3) of thymic carcinoma patients and 81% (26 of 32) of thymoma patients. PD-1 expression was detected in 77% (27 of 35), including 33% (1 of 3) of thymic carcinoma patients and 81% (26 of 32) thymoma patients. High PD-1 expression was associated with lower grade tumors. Unlike prior studies, PD-L1 expression was not associated with higher grade tumors or higher stage. Neither PD-L1 nor PD-1 expression was significantly associated with survival. Three patients with thymoma had multiple tumor sections evaluated for expression of PD-1/L1, with differing expression patterns of both PD-L1 and PD-1 observed in two patients., Conclusions: This study confirms high expression of PD-L1 and PD-1 in TET and shows for the first time intratumoral heterogeneity of PD-L1 and PD-1 in thymoma patients., (Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Programmed death ligand 1 (PD-L1), an immune check point inhibitor, is known to be expressed in several malignancies and is being considered as a prognostic factor and a potential immunotherapeutic target. The aim of this study was to characterize PD-L1 expression in thymomas and to determine correlation with clinicopathological features and previously published studies in the literature., Methods: Tissue microarrays were prepared from selected blocks of thymomas and immunohistochemistry (IHC) for PD-L1 was performed. Cases were considered as PD-L1 positive or negative depending on whether the percentage of stained thymic epithelial cells were <25 or >25%. Results were compared clinically and with previously published studies using Google and Pubmed search engines., Results: Of 84 cases of thymoma, 69 (82.1%) revealed PD-L1 positivity in >25% cells. 94.23% of type B thymoma subtypes (B1/B2/B3) were PD-L1 positive (P < 0.001). There was no correlation of PD-L1 with age, gender, myasthenia gravis, the tumor size or stage of disease. Nine studies were available in the literature; most of which showed PD-L1 expression in higher stage and B subtype however percentage positivity varied from 53.7% to over 90%., Conclusions: PD-L1 expression is frequent in type B (B1/B2/B3) thymomas. It can be easily evaluated by IHC even on small biopsies in unresectable cases, thereby enabling improved clinical evaluation as well as prognostic stratification of patients. It will serve as a potential indicator for benefit from anti-PD-L1 antibody immunotherapy in thymomas., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Sixteen cases of primary thymic adenocarcinoma are presented. The patients are 9 men and 7 women between the ages of 22 and 68 years (average, 45 years) who presented with non-specific symptoms including cough, chest pain, and dyspnea. Diagnostic imaging revealed the presence of an anterior mediastinal mass, which was surgically removed in all of the patients. Histologically, none of the tumors was encapsulated and showed different growth patterns including mucinous, non-mucinous, and papillary features. The majority of cases showed mixed growth pattern, and the tumor was limited to the mediastinum with only a few cases extending to lymph nodes or pericardium. In two cases, the adenocarcinoma was associated with a thymoma. Immunohistochemical stains were performed, and their positive staining varied depending on the histology of the tumors, showing positive staining in some cases for keratin 7, keratin 20, CEA, CD5, CD117, and CDX-2. PAX8 and TTF-1 were negative in all the tumors. Follow-up information was obtained in 10 patients over a period of 1 to 12 years, indicating that three patients had died within a period of 14 months, one with brain metastasis, while seven patients have remained alive without recurrence. The cases herein described span the spectrum of thymic epithelial tumors and highlight the importance of recognizing this particular type of carcinoma, as it may follow a different outcome and require different treatment options., (Copyright © 2018. Published by Elsevier Inc.)